Bone invigorates metstatic seeding [evolving barcode]

Following the removal of implanted mammary tumors, nude mice develop multiple-organ metastases at late stage. The metastases may originate from the primary tumors before the resection surgery, or alternatively, from some established metastases. By multiple approaches, we have proved that bone environment could invigorate cancer cells for further dissemination. this study aims to examine if metastatic dissemination from bone to other sites occurs in natural setting of metastatic spread. We herein apply the rapidly evolving barcode system using homing guide RNA/Cas9 to trace the metastases formation in mouse. hgRNA/Cas9 is a self-targeting Crispr system which allows the mutation occurs in the DNA sequence of guide RNA. Tumor cells wer labelled with doxycycline inducible evolving barcoding system. Upon doxycycline treatment the DNA sequence of hgRNA accumulate mutations with time. The diversity of barcodes in each lesion can infer the timeing of seeding while the mutation patterns of barcodes suggest the phylogenetic correlation of metastases. Several findings were made on this study. First, at the terminal stage, multi-organ metastases are not genetically grouped according to sites of metastases. Nonnegative Matrix Factorization (NMF) analysis of mutant barcodes suggested the early disseminated metastases, which have highest level of Shannon entropy, were featured with a common cluster of mutant barcodes irrespective of their locations. Second, most metastases are potentially multiclonal as indicated by multiple clusters of independent mutant barcodes. Third, when we use Shannon entropy as an index of metastasis age , putative parent-child relationship between metastases with unique mutant barcodes clearly exemplified secondary metastatic seeding from bone to other organs. Finally, we did not observe a clear correlation between tumor burden and Shannon entropy across different metastases, suggesting that putative parental metastases might remain small after seeding further metastases. Nude mice or C57BL/6 mice were implanted with barcoded MDA-MB-231 or AT-3 cells, respectively. the mammary tumors were removed when the tumor size reached 1.2cm, and a dose of doxycycline were administated to the mice weekly via intra-peritoneal injection for 5 weeks in nude mice or 4 weeks in C57-BL/6 mice. the metastatic lesions were then dissected with assistance of ex vivo BLI imaging and the DNA of each metastases were isolated. The barcodes from DNA samples were then targeted sequenced using Illumina NextSeq 500/550. Mutation events were categorized from the sequence of barcodes and subjected to the following bioinformatic analysis.